The molecular mechanism of phototransduction and of light dependent retinal degeneration will be studied by an interdisciplinary approach using functional assays applied to Drosophila visual mutants. Retinal degeneration, mobilization of Ca2+ and the turn-off of the light activated photopigment will be investigated. Our hypothesis suggests that a deficiency in a protein phosphatase which no longer can cope with the light-activated protein kinase C (PKC) is the primary lesion in the retinal degeneration B (rdgB) Drosophila mutant. This results in an unbalanced regulation of voltage and phosphorylation dependent Ca2+ channels leading to toxic [Ca2+]i levels and thereby to photoreceptor cell death. The consequences of deficient protein phosphatase in the mutant will be studied in vivo using pulse labeling with 32pi and visualization of the phosphoproteins by SDS-PAGE and autoradiography. Identification of the deficient phosphatase as Ca2+ calmodulin dependent enzyme (Calcineurin) will be tested by in vitro assay using a specific synthetic peptide and potent and selective peptide inhibitors of calmodulin. The properties of the regenerative Ca2+ spikes, a characteristic of retinal degeneration in the rdgB mutant will be studied using isolated ommatidia and optical measurements of [Ca2+]i. Voltage dependent Ca2+ channel blockers will be tested for their efficacy as therapeutic means for prevention of light and chemically-induced retinal degeneration. The Drosophila trp mutant and the Lucilia nss mutant will be used to verify the hypothesis that the product of the recently cloned trp gene is a new type of Ca2+ transporter located at the plasma membrane acting to replenish the intracellular Ca2+ store, a function needed for excitation. The effect of La3+ in turning the wild type response into a trp phenotype will be investigated using current measurements from isolated ommatidia with a suction pipette and by optical methods. Cytolocalization with trp antibodies and reconstitution of the trp protein into phospholipid vesicles will test its function in Ca2+ transport. The turn off of the active photopigment will be investigated by analysis of the prolong depolarizing afterpotential (PDA). The hypothesis that the PDA is caused by persistently active photopigment molecules will be tested. Two Drosophila mutants, a rhodopsin mutant lacking the phosphorylation sites at the C-terminus and a mutant lacking PKC activity will be used. Correlation of the PDA monitored by GTPase and phosphorylation of rhodopsin under PDA and non-PDA conditions will be investigated. Analysis of rhodopsin phosphopeptide map in wild type and the mutant flies will test the relevance of particular phosphorylation sites to inactivation.